Abstract
The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical cross-linking using mRNA analogues that contain 4-thiouridine (s4U) or s4U modified with azidophenylacyl bromide (APAB), either two nucleotides upstream or eight nucleotides downstream from the nucleotide sequence ACC, the codon for tRNA(Thr). The sequences of the mRNA analogues were described earlier (Stade, K., Rinke-Appel, J., and Brimacombe, R. (1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe, R. (1991) EMBO J. 10, 2195-2202). Under equilibrium conditions, both of these mRNA analogues bind and cross-link to 70 S ribosomes without the presence of tRNA(Thr); however, there are significant increases both in binding and particularly in cross-linking in the presence of the tRNA(Thr). Four regions contain cross-linking sites that increase in the presence of tRNA, C1395, A532, A1196 (and minor sites around these three positions), and C1533/U1532. Three other cross-linking sites, U723, A845, and U1381, show very little change in extent of cross-linking when tRNA is present. A conformational change in the 30 S subunit allowing additional accessibility to the 16 S rRNA by the mRNA analogues upon tRNA binding best explains the behavior of the tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.
Highlights
If the numbering of positions in the mRNA analogue are made from "+ 1" being the first nucleotide of the triplet used in the P-site, these are +6 in the mRNA to U1052 in the 16 S rRNA, +7 in the mRNA to C1395 in the 16 S rRNA, and + 11 in the mRNA to A532 in the 16 S rRNA (15)
To test if the presence of the two types ofmRNA binding sites reflected ribosome heterogeneity, tRNA binding was measured at an mRNA analogue concentration that resulted in an average of 1 mRNA analogue/ribosome. tRNA binding was found to be at least 0.85 tRNNribosome, all confined to the P-site
We do not detect U 1052 but in stead det ect A1196 whi ch is very close to U 1052 in th e seconda ry st ruct ure; th ese pr obabl y represen t th e same mRNA -16 S rRNA inte ra ction region
Summary
Extent of cross- linking to 16 S rRNA a nd 23 S rRNA is indi cated as per cent mol mRNA a na logue/mol rRNA species. Entries indicate the normalized relative intensity of bands for cross-linked versus control samples. The two values for each are cross-linking without and with tRNAThr. For all cross-linking sites, it was assumed that reverse transcriptase stops completely at the 3'-nucleotide to the cross-link. Positions that had an increase in relative band intensity values of at least 1.5 either without or with tRNA are listed. Empty areas indicate no cross-links under any condition. The most frequent cross-linking site in each group is underlined. C13 89 1.4, 1.6 V 13 9 0 22 32 V 13 9 1 1:9: 3:1 G13 9 2 2.1, 3.9 V 13 9 3 14 19 C1395 5."6: 9."3
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